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Phosphoproteomics of MAPK Inhibition in BRAF-Mutated Cells and a Role for the Lethal Synergism of Dual BRAF and CK2 Inhibition

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Phosphoproteomics of MAPK Inhibition in BRAF-Mutated Cells and a Role for the Lethal Synergism of Dual BRAF and CK2 Inhibition Published OnlineFirst May 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0938 Molecular Cancer Cancer Biology and Signal Transduction Therapeutics Phosphoproteomics of MAPK Inhibition in BRAF-Mutated Cells and a Role for the Lethal Synergism of Dual BRAF and CK2 Inhibition Robert Parker1, Roderick Clifton-Bligh2, and Mark P. Molloy1 Abstract Activating mutations in the MAPK pathway are prevalent drivers of several cancers. The chief consequence of these mutations is a hyperactive ERK1/2 MAPK able to promote cell proliferation, producing a critical hallmark of metastatic disease. The biochemistry of the ERK pathway is well characterized; however, how the pathway achieves different outcomes in the face of genetic aberrations of cancer and subsequent treatment with chemical inhibitors is not clear. To investigate this, we used mass spectrometry to complete a global phosphoproteomic analysis of a BRAFV600E thyroid cancer cell line (SW1736) after treatment with the mutation-selective inhibitor vemurafenib (PLX4032) and MEK1/2 inhibitor selumetinib (AZD6244). We identified thousands of phosphorylation events orchestrated in BRAFV600E cells and performed kinase landscape analysis to identify putative kinases regulated in response to MAPK blockade. The abundance of phosphopeptides containing consensus motifs for acidophilic kinases increased after short-term inhibition with these compounds. We showed that coinhibition of the pleiotropic acidophilic protein kinase CK2 (CK2) and BRAFV600E synergistically reduced proliferation in patient-derived melanomas and thyroid cancer cells harboring the BRAF lesion. We investigated this mechanism and show a role for CK2 in controlling AKT activation that was not reliant on changes to PTEN or PDK1 phosphorylation. These findings highlight a role for CK2 blockade in potentiating the antiproliferative effects of BRAF and MEK inhibition in BRAF cancers. Mol Cancer Ther; 13(7); 1–13. Ó2014 AACR. Introduction kinase able to hyper-phosphorylate MAPK kinases MEK1/ Aberrant activity of protein kinases drives many of the 2, driving cell transformation through unrestrained ERK1/ hallmarks of cancer and also participates in the develop- 2 activation. The discovery of specific activating mutations ment of resistance to current treatments (1). In many has led to the clinical development of mutation-specific, cancers, dysregulation of the MAPK pathway is associated small-molecule kinase inhibitors. In melanoma, one such with poor prognosis, which results from activating muta- compound, vemurafenib (PLX4032), has shown high effi- tions in genes encoding cytosolic signaling proteins (e.g., cacy in BRAFV600E-positive patients (5), and the logical BRAF and RAS) or receptor tyrosine kinases (e.g., EGFR application of this approach in thyroid cancer is under and RET). The BRAF gene is found mutated in approxi- investigation. However, as with many targeted therapies, mately 1 of 4 of all anaplastic thyroid carcinomas (ATC), acquired resistance to treatment is common, and thus and 40% to 60% of papillary thyroid carcinomas (PTC; successful application of targeted therapies will benefit refs. 2–4). BRAF is commonly mutated by a single transver- from more sophisticated understanding of the events con- sion (T1799A) that codes for a missense protein (V600E). trolled by oncogenic mutations and the molecular Biochemically, BRAFV600E mimics phosphorylation of the responses that result from inhibiting these enzymes. T598 and S601 residues producing a constitutively active The impact of pharmacologic blocking of BRAFV600E by selective inhibition in thyroid cancer has been dem- onstrated in vivo. Inhibition reduces cell proliferation in Authors' Affiliations: 1Australian Proteome Analysis Facility, Department PTC/ATC mouse tumor xenograft (6) and recently of Chemistry and Biomolecular Sciences, Macquarie University; and 2Kol- ling Institute of Medical Research, University of Sydney, Sydney, New vemurafenib was shown to suppress tumor growth in South Wales, Australia BRAFV600E human ATC (7). At a molecular level, kb Note: Supplementary data for this article are available at Molecular Cancer BRAFV600E mediates activation of the NF- transcrip- Therapeutics Online (http://mct.aacrjournals.org/). tion factor, epigenetic reprogramming through methyla- TIMP3 HMGB2 Corresponding Author: Mark P. Molloy, Macquarie University, Research tion, and/or expression of genes such as , , Park Drive, Sydney, NSW 2109, Australia. Phone: 612-9850-6218; Fax: metalloproteases, and other structural extracellular 612-9850-6200; E-mail: [email protected] matrix genes that can promote proliferation and cell doi: 10.1158/1535-7163.MCT-13-0938 invasion (8–10). NF-kb–driven expression of TIMP1 is Ó2014 American Association for Cancer Research. also implicated in BRAFV600E thyroid cancers, able to www.aacrjournals.org OF1 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst May 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0938 Parker et al. activate the PI3K/AKT pathway sustaining cell prolifer- tive inhibitor; 2 mmol/L vemurafenib (PLX4032; Selleck- ation, with one possible consequence being the over chem) or 1 to 2 mmol/L selumetinib (AZD-6244; Sell- activation of the mTOR pathway (8, 11). Gene expression, eckchem) or DMSO (Sigma) for 30 minutes before cell methylation, and molecular studies have revealed several lysis. Three independent cell cultures were used per cell key processes regulated by BRAF in thyroid cancer, but line and condition. Cells were lysed as described in (12) currently little is known about dysregulated posttransla- with minor modifications (see Supplementary Methods) tional control of protein signaling. In particular, little is and protein amounts determined. A total of 1 to 25 pmol of known about how selective inhibition of BRAFV600E siRNA-targeting transcripts of the human CK2a (Cell alters the output of cell signaling processes. Data outlining Signaling) and nontarget control siRNA were used to the signaling events regulated by BRAFV600E, and how knock down CK2 expression in melanoma cells. siRNA cells respond to small-molecule–based inhibition, are was delivered using Lipofectamine RNAiMAX for 72 vital to designing combination therapy programs that hours, protein levels were examined by Western blot are effective and combat the development of acquired analysis. resistance. Lack of knowledge of phosphorylated sites within Protein digestion and phosphopeptide enrichment proteins and identification of upstream regulating kinases A total of 500 mg of proteins were reduced, alkylated, are major limiting factors in understanding what path- and digested with trypsin (Promega) overnight at 37C. ways are activated in cancer. Currently, several reliable Samples were acidified, adjusted to 80 mg/mL glycolic techniques for phosphopeptide enrichment and charac- acid, and phosphopeptides purified by the addition of m terization using mass spectrometry have been developed 5 mg of TiO2 beads (Titansphere, 10 m) for 1 hour. Beads and promise to add significantly to this knowledge base were extensively washed and phosphopeptides were (12, 13). Temporal characterization of the phosphopro- eluted with consecutive 100 mL additions of 1% (v/v) teome cellular landscape provides a direct readout ammonia (Sigma) with 0%, 30%, and 50% (v/v) acetoni- of kinase substrates and can be used to develop hypoth- trile. Samples were immediately dried and resuspended eses about likely active kinases under those conditions. in 1% (v/v) trifluoroacetic acid, 5% (v/v) acetonitrile for Here, we used established techniques to profile the LC/MS-MS. phosphoproteome of a drug-sensitive BRAFV600E-pos- itive ATC cell line (SW-1736), and monitor the quanti- Mass spectrometry (LC-MS/MS) tative response of approximately 2,000 phosphosites Samples were analyzed by LC-MS/MS using a Triple- after selective blocking of BRAFV600E and MEK1/2 TOF 5600 mass spectrometer (AB Sciex) and peptides with the clinically tested small-molecule inhibitors identified using ProteinPilot v4.5 and Mascot v2.2 as vemurafenib (PLX4032) and selumetinib (AZD6244). described in Supplementary Methods. Label-free quanti- We used thyroid cancer cell lines with defined muta- tation (LFQ) using extracted ion chromatograms was con- tions in BRAF and RET to demonstrate mutation-specific ducted using Skyline (14) and statistical analysis carried response in cell growth, cell cycle, and phosphorylation of out using the DanteR scripts. Parallel reaction monitoring known and novel effectors. Motif analysis of the flanking was conducted on the TripleTOF 5600 and analyzed using linear amino acid sequences of regulated phosphosites Skyline as described in Supplementary Methods. revealed several regulatory kinases that function in BRAFV600E signaling and in response to inhibition. Phosphosite localization and kinase assignment Although ERK1/2 and cyclin dependent kinase (CDK1/ To localize modifications, search results were processed 2) substrates were expected in downregulated sites after using Scaffold PTM (ProteomeSoftware), which uses A- BRAF inhibition, we also observed increases in some score (15) and a probability function to assign confidence phosphosubstrates consistent with the activity of acido- to amino acid modification location based on available philic kinase(s). We demonstrated that combinatorial inhi- peak depth present in MS/MS spectra. Upstream kinases bition of BRAF and the acidophilic kinase CK2 in thyroid were putatively assigned using the
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